Biological activities of sweet potato (Ipomoea batatas L.) tips and tubers

Abstract This study was conducted to evaluate the biological activities of sweet potato tips and tubers. Antioxidant activity of 2,2‐azino‐bis 93‐ethlbenzothiazoline‐6‐sulphonic acid) diammonium salt (ABTS) and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activities had the highest value of 32.45 mg, AAE/g, and 15.10 mg AAE/g, respectively, in ‘Pungwonmi’ tips. Angiotensin converting enzyme I inhibitory activity ranged between 47.72% in ‘Sinjami’ tubers and 62.25% in ‘Pungwonmi’ tips. α‐Glucosidase inhibitory activity had the highest value of 78.81% and 62.93% in ‘Pungwonmi’ tips and ‘Juhwangmi’ tubers, respectively. In particular, ‘Pungwonmi’ tips had the most effective inhibiting effect on intracellular reactive oxygen species levels in HepG2 cells. Wound healing assay result revealed that ‘Sinjami’ showed 75% wound healing effect. For skin whitening, ‘Pungwonmi’ tips showed 63% activity at 10 mg/ml. These results suggest that sweet potato tips and tubers can be used to develop functional food and cosmetic materials.

For skin whitening, 'Pungwonmi' tips showed 63% activity at 10 mg/ml. These results suggest that sweet potato tips and tubers can be used to develop functional food and cosmetic materials.

K E Y W O R D S
antioxidant, biological activities, sweet potato tips, sweet potato tubers [corrections added on 30 July 2022, 6th author's name was corrected from Uun-Bae Kim to Yun- Bae Kim] are edible. Owing to the high content of polyphenols in the tips, it is used in the manufacturing of various foods in foreign countries by adding the dried powdered tips to dough for bread, ice cream, juice, and tea (Islam, 2006). In Korea, the leaves and stalks of sweet potato have been used as herbs or for soups for a long time, however, the tips are often discarded or used only as feed owing to its insufficient availability (Li et al., 2012). Sweet potato tips are not only rich in water-soluble dietary fiber but also rich in protein compared to other vegetables. They are also rich in iron; potassium; vitamin B2, C, and E; carotene (Ishida et al., 2000); phenolic compounds; anthocyanins; and caffeic acid. They are known to also contain many antioxidants, including phosphoric acid derivatives (Islam et al., 2003).
Sweet potatoes are edible crops with both leaves, stems, and roots, and are highly economical and productive. In particular, sweet potato leaves and stems are known to have better antioxidant and phenolic components than regular vegetables. However, leaves and tips other than stems are treated as byproducts and are discarded or used as livestock feed. Therefore, in this study, functional ingredients and physiological activity were evaluated and their importance was verified in order to increase the availability of sweet potato tips and tubers and use them as various functional food materials.

| ABTS and DPPH radical scavenging activity
ABTS and DPPH radical scavenging abilities were measured using the method described by Choi et al. (2006), with some modifications. After leaving the ABTS (7.4 mM) and potassium persulfate (2.6 mM) in the dark for one day to form ABTS. + cations so that the absorbance value is 1.4 at 735 nm, it was diluted with distilled water. To 1 ml of the diluted ABTS. + solution, 50 μl of the extract was dissolved in each concentration, and the change in absorbance was measured after exactly 30 min. The ABTS radical scavenging ability was expressed as the equivalent of ascorbic acid. DPPH radical scavenging activity was used after dissolving 0.00788 g of 0.2 mM DPPH in 99.9% ethanol and adjusted to 100 ml for 60 min. After adding 0.2 ml of the sample to 0.8 ml of the DPPH solution, the absorbance was measured at 520 nm by adding 0.2 ml of the sample at ambient temperature for 30 min, and the radical scavenging ability was expressed as an IC 50 value.

| Angiotensin converting enzyme I inhibitory activity
Angiotensin converting enzyme (ACE) inhibitory activity for each extract was measured using the method described by Kwon et al. (2006) with little modification. ACE inhibitory activity was measured using 5 mM HHL (Hippuryl-His-Leu) substrate and dissolved in 0.1 M potassium phosphate buffer (pH 8.3) containing 0.3 M NaCl, and the 0.2 mU ACE purifying enzyme was dissolved in 0.1 M potassium phosphate buffer (pH 7.0) and used. Eighty microliter of 0.2 mU ACE purified enzyme solution and 100 μl of 5 mM HHL substrate was added to 100 μl of the extract, and then left at 37°C for 60 min. To stop the reaction, 250 μl of 1 M HCl was added and then filtered through 0.45 μm syringe filters, and the resultant sample was then analyzed using a HPLC (ACME 9000 system, Younglin, Anyang, Korea), and the ACE inhibition rate (%) was calculated. The column was a C-18 column (Mightysil RP-18 GP column, 4.6 × 250 mm, Kanto Chemical, Tokyo, Japan), the mobile phase A was 10 mM phosphoric acid (pH 2.5), and the mobile phase B was a methanol. The initial ratio of A and B started at 100:0, followed by a stepwise gradient system with a ratio of 40:60 at 12 min, 0:100 at 19 min, and 100:0 at 25 min. The flow rate was 0.8 ml/min, the injection volume was 20 μl, and the UV detector (228 nm) was used for detection.

| α-Glucosidase inhibitory activity
α-Glucosidase inhibitory activity was measured using the method described by Tibbot and Skadsen (1996). α-Glucosidase (0.35 U/ml) and ρ-NPG (1.5 mM ρ-nitrophenylα-D-glucopyranoside) were dissolved in 0.1 M sodium phosphate buffer (pH 7.0), and 50 μl of each extract was used at 0.35 unit. The mixture was mixed with 100 μl of α-glucosidase enzyme solution, preincubated at 37°C for 10 min, and then 50 μl of 1.5 mM ρ-NPG was added at 37°C for 20 min. After stopping the reaction by adding 1 ml of 1 M Na 2 CO 3 , absorbance was measured at 405 nm using a UV spectrophotometer (Epoch microplate spectrophotometer, Biotek Instruments, Vermont, USA), and the inhibition rate (%) was calculated. Acarbose (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control.

| Reactive oxygen species inhibitory effects in HepG2 cells
The human liver cancer cell line (HepG2 human liver cancer cell line) used in this experiment was purchased from the Korea Cell Line Bank (Seoul, Korea). The cytotoxicity of the extract was measured using the MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay according to the method described by Ishiyama et al. (1996). reactive oxygen species (ROS) was measured using the fluorescent probe DCFH-DA method. Cells were dispensed at a concentration of 5 × 10 4 cells/well into a 96-well plate (100 μl) and cultured at 37°C in a 5% CO 2 incubator for 24 h. The extract was diluted with FBS-free DMEM medium at a concentration of 100 μg/ ml. The medium used for the culture was removed, and the diluted sample was added to the medium. After pretreating the sample for 24 h, 250 μM DCFH-DA was added to each well for 1 h at 37°C.
After washing the cells with PBS, 1 mM tert-butyl hydroperoxide (TBHP) was added. The corresponding ROS between cells were measured using a fluorescent spectrophotometer (Perkin-Elmer, Norwalk, CT, USA) for 120 min at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

| Wound healing assay for APRE-19 cell
ARPE-19 cells were inoculated in a 24-well plate at a concentration of 2 × 10 5 cells/well. DMEM-F12 (0% FBS) was used as the medium and a stabilization time of 7 h was set. The cells were observed under a microscope, and where the bottom of the 24-well plate was not visible, scratch was created by scratching the bottom with a yellow tip. The existing culture solution was removed and replaced with a new culture solution (0% FBS in DMEM/F12). After the extract was treated with 10 μl per well so that the final concentration was 32 and 100 μg/ml, pictures of each well were taken after incubation at 37°C and 5% CO 2 incubator. After scratching, the condition of each well was checked at 18 h and a picture was taken. After organizing the photographic data, the area of the scratch for each photograph taken was measured using the Image studio program, and the rate of wound healing was calculated.

| Tyrosinase inhibitory activity
Tyrosinase inhibitory activity was measured according to the method described by Yagi et al. (1987). In the reaction zone, 0.2 ml of mushroom tyrosinase (110 U/ml) was added to 0.5 ml of 0.175 M sodium phosphate buffer (pH 6.8), 0.2 ml of 10 mM L-DOPA, and 0.1 ml of sample solution and reacted at 37°C for 2 min. The DOPA chrome generated in the reaction solution was measured at a wavelength of 475 nm using a UV spectrophotometer (Epoch microplate spectrophotometer, Biotek Instruments, Vermont, USA).
The tyrosinase inhibitory activity was expressed as the rate of decrease in absorbance of the sample solution (with and without addition).

| Statistical analysis
For statistical analysis, the mean and standard deviation of each measurement group was calculated using the SPSS statistical program (Statistical Package for the Social Science, Ver. 18.0, SPSS Inc., Chicago, IL, USA), and the difference between the mean of the measured values was independent. A sample t-test (Student's t-test) was performed, and a one-way ANOVA was performed to determine whether there was a difference between treatment conditions.

| ABTS and DPPH radical scavenging activity
The results of the analysis of ABTS and DPPH radical scavenging activity of sweet potato tip and tuber extracts are shown in Table 1. ABTS radical scavenging activity was in the range of 2.68-32.45 mg AAE/g, and was 32.45 mg AAE/g for Pungwonmi tips, which was the most active. The DPPH radical scavenging activity was in the range of 0.53-15.10 mg AAE/g, and was 15.10 mg AAE/g for Pungwonmi tips. Both ABTS and DPPH radical scavenging activity were higher in the tip than the tubers. The antioxidant activity of tuber was higher in Sinjami than that of Juhwangmi (p < .05), and the Pungwonmi tips was significantly higher than that of the other two cultivar (p < .05), and there was no significant difference between Hayanmi and Juhwangmi tips. According to a study by Lee, Park, et al. (2007), the high phenolic substances in sweet potato parts and their antioxidant activity corroborate the high content of phenolic substances and good antioxidant activity Inhibitory activity( % ) = (1 − absorbance at the sample addition ∕ absorbance at no addition) × 100 TA B L E 1 ABTS and DPPH radical scavenging activity of different sweet potato cultivars.
in leaves and tips in the present study. According to a study by Li et al. (2012), the ABTS radical scavenging activity of sweet potato leaves and stalks was in the range of 8.02-14.95 AAE/g, and the content of the leaves was higher than that of the stalks. In addition, according to a study by Woo et al. (2012), the ABTS and DPPH radical scavenging activity of sweet potato tubers were 4.38-6.63 and 5.46-9.58 mg/g, respectively, and are similar to the results of this study.

| ACE I inhibitory activity
The results of ACE inhibitory activity of sweet potato tip and tuber extracts are shown in Figure 1. ACE inhibitory activity significantly increased with increasing concentration in all cultivars, and showed 47.72%-62.25% activity at 10 mg/ml. ACE inhibitory activity was highest at the Pungwonmi tips and showed a high activity of 22.06% at 1 mg/ml, 42.05% at 5 mg/ml, and 62.25% at 10 mg/ml. In the study by , which analyzed the antihypertensive activity of each sweet potato part, the ACE inhibitory activity was 1.5 times higher in the tips than in the tubers, showing a trend that is consistent with this study. As a result of statistical analysis of ACE inhibitory activity, there was a significant difference between tip and tuber at a concentration of 10 mg/ml (p < .05). In the tip, Pungwonmi cultivar was the highest, followed by Juhwangmi and Hayanmi cultivar (p < .05). However, there was no significant difference between cultivars in the tubers. In addition, a study by , which analyzed the ACE inhibitory activity of sweet potato tips and tubers, showed excellent antihypertensive activity that was 1.9-3.7 times higher than that of perilla leaves, bean sprouts, and spinach. Enalapril, a treatment material for hypertension, was used as a positive control, and it showed 98.80% activity at 1 mg/ml. Studies showing the ACE inhibitory activity of herb plants, medicinal plants, and onion seasonings have been reported (Kwon et al., 2006;Lee et al., 2004).

| α-Glucosidase inhibitory activity
The results of the antidiabetic activity of sweet potato tip and tuber extracts are shown in Figure 2. Antidiabetic activity significantly increased with increasing concentration of the extracts in all cultivars, and showed an inhibitory activity in the range of 36.17%-69.57% at 1 mg/ml. Acarbose, an antidiabetic treatment, which was used as a positive control in the experiment, showed 84.49% activity at 1 mg/ml. Antidiabetic activity was as high as 69.57% for Pungwonmi tips and 82% for acarbose. In a study by Matsui et al. (2004), it was reported that 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid had excellent inhibitory effects on maltase and 3,4,5-triCQA in diabetes model mice. When administered orally, the blood sugar content was significantly reduced.

| ROS inhibitory effects in HepG2 cells
The results of ROS inhibitory effects are shown in Figure 3. From the result of the ROS inhibition activity test using hepatocytes,

ROS production increased in the cells induced oxidative stress
with TBHP, compared to normal cells, however, the ROS production was inhibited in a concentration-dependent manner in cells treated with sweet potato tips and tubers extracts. Particularly, it was found that Pungwonmi tips were effective in inhibiting ROS production. When oxidative stress was induced with TBHP in HepG2 cells, ROS production increased to 221.88% compared to that of the control, however, ROS production was suppressed by

| Wound healing assay for APRE-19 cell
The results of wound healing assay of sweet potato tip and tuber extracts are shown in Figure 4. Result of the Image J program analysis revealed that Sinjami tubers showed 75% wound healing effect in the 100 μg/ml treatment group. In the 32 μg/ml treatment group, Sinjami tubers showed 90% wound healing effect. Sinjami tubers are a representative of purple sweet potato cultivar with high anthocyanin content . Anthocyanins are reported to have antioxidant, antibacterial, hepatoprotective, and antihypertensive effects (Kang et al., 2003;Qi et al., 2005), and are known to be effective in treating eye disease (Kang et al., 2019). In a study by Kang et al. (2019), anthocyanin reduced the inflammatory response on the surface of the eye; they also reported its effect in improving the symptoms of dry eye syndrome, similar to the result of a study by Riva et al. (2017).

| Tyrosinase inhibitory activity
The results of the tyrosinase inhibitory activity of sweet potato tips and tubers are shown in Figure 5. The positive control, F I G U R E 2 α-Glucosidase inhibitory activity of different sweet potato cultivar. The concentration of positive control was 1 mg/ml. Data are expressed as mean ± SD. Different small letters in the same items indicate a significant difference among different cultivar by duncan's range test (p < .05).  Lee et al. (2006) also confirmed the effect of tyrosinase inhibitory activity of sweet potato extract, and it was reported that the tyrosinase inhibitory activity increased as the concentration of the sweet potato extract increased.

| CON CLUS ION
This study evaluated the biological activities of antihypertension, antidiabetes, hepatoprotective, antioxidant, eye function, and skin whitening effects of sweet potato tips and tubers to develop the functional food and cosmetic materials. As a result of ABTS and DPPH radical scavenging activity, 'Pungwonmi' tips showed the highest activity. The results of ACE inhibitory activity for antihypertensive activity was also highest in 'Pungwonmi' tips. α-Glucosidase and tyrosinase inhibitory activity increased significantly with increasing concentration in all cultivars; the activity was higher at the tips than the tubers. α-Glucosidase inhibitory activity showed the highest activity of 69.57% at 10 mg/ml in 'Pungwonmi' tips. When oxidative stress was induced with TBHP in HepG2 cells, ROS production increased by 221.88% compared to that in the control, however the 'Pungwonmi' tips decreased by 102%. In the wound healing assay for ARPE-19 cell experiment, the effect of creating a scratch on the ARPE-19 cell was highest in the 'Sinjami' tubers. These results suggest that, sweet potato tips and tubers are excellent in improving eye disease, have whitening effects, and can be used as functional food and cosmetic material.

ACK N OWLED G EM ENTS
This thesis is the result of the Rural Development Administration's (Task number: PJ0142332020) project research fund support, whom the authors thank for the research fund support.

FU N D I N G I N FO R M ATI O N
Rural Development Administration (RDA), Republic of Korea, Grant number: PJ0142332020

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.